Capillary electrophoresis ("CE") is a well known separation technique that is of increasing interest to those concerned with separations. It is a modification of electrophoresis, typically practiced in a thin glass capillary instead of on a 2-dimensional surface such as paper or in a gel. This technique offers the benefits of high efficiency and resolution, rapid separations, the ability to analyze small sample amounts, and a desirable simplicity from the point of view of the apparatus required when compared to competing analytical techniques such as gel electrophoresis, gas chromatography, and liquid chromatography. As in all separation systems high resolution is the end objective and as in other systems resolution is a function of efficiency (theoretical plates) and selectivity (Weinberger, R. "Practical Capillary Electrophoresis", Academic Press, San Diego, Calif. 1993).
The benefits of capillary electrophoresis derive to a large extent from the use of narrow diameter capillary tubes, which permit efficient removal of the heat generated in the separation process. This heat removal prevents convective mixing which would degrade the separating power. The narrow diameter tubes also allow high voltages to be used to generate the electric field in the capillary while limiting current flow and hence heat generation.
A CE separation begins by filling the capillary with a supporting electrolyte. Next, a small amount of sample is injected into one end of the capillary. Typical sample injection volumes range from 1-20 nanoliters. After sample injection, a high voltage is applied to the capillary and the sample components are separated on the basis of different charge/mass ratios. A capillary electrophoretic separation can also be augmented with a bulk fluid flow, called electroosmotic flow. If present, it moves all components through the capillary tube at the same rate, and generally does not contribute to the resolution of different sample components. Eventually, the sample components move through an appropriate detector, such as a UV detector. This can provide detection and quantitation of each separated sample zone.
A micelle is a colloidal particle formed from surfactant molecules. The practice of capillary electrophoresis in the presence of micelles is commonly referred to as micellar electrokinetic chromatography (Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A. and Ando, T. Analytical Chemistry, 1984, (56) 111-113; Terabe, S., Otsuka, K., and Ando, T. Analytical Chemistry, 1985, (57)834-841). This term will be used to refer to both micellar electrophoretic separations, (separations where the electroosmotic flow is negligible), and micellar electrokinetic separation (separations where the electroosmotic flow impacts the separation time). The equations for retention and resolution in MEKC are shown as equations 1 and 2 (Terabe, S., et al. supra). ##EQU1## where t.sub.r =retention time of the solute t.sub.o =retention time of solute in the absence of micelles
t.sub.mc =micelle retention time
and k is the solute's capacity factor. ##EQU2## where N=efficiency (theoretical plates) .alpha.=selectivity.
Equation 3 is the resolution equation for HPLC, with all terms as previously defined. ##EQU3##
The resolution equation for MEKC is very similar to the resolution equation for HPLC. In fact, as t.sub.mc approaches infinity, the equations become identical (equation 3). Considering the case where t.sub.mc or t.sub.o equals infinity, one can easily se the difference between HPLC and MEKC. The practical difference between HPLC and MEKC is related to the efficiency term of the resolution equation. In HPLC a typical value of N (theoretical plates) is 5000 while a typical value of N for MEKC is 100,000. In quantitative terms a widely accepted goal for resolution of two peaks is a value of 1.5. Assuming k'=1, then the resolution requirement of 1.5 requires an alpha value of 1.20 for the HPLC case. However, the much higher efficiency of MKC determines that a much smaller alpha of 1.04 achieves the same resolution of 1.5. Thus, if the small alphas associated with many partial HPLC separations could be achieved in an MEKC system, useful resolutions would result. (Note that because resolution depends on the term ((.alpha.-1)/.alpha.), an alpha of 1.04 provides twice the resolution of an alpha of 1.02).
Chiral separations have been accomplished using a variety of techniques. Over the last thirty years investigators have shown that chiral separations are possible using gas chromatography (GC) and liquid chromatography (LC) (Zief, M. and Crane, L. J., Editors, "Chromatographic Chiral Separations" Marcel Dekker, Inc., New York. Basel, 1988), gel electrophoresis (Barton, J. K., J. Biomolecular Structure and Dynamics, 1983, (1) 621-632), paper electrophoresis (Fanali, S., Cardaci V., Ossicini, L., J. Chromatogr. 1983, (165) 131-135) and capillary electrophoresis (CE) (Snopek, J., Jelinek, I. and Smolkova-Keulemansova, E. Journal of Chromatography, 1992, (609) 1-17). These separations are based on the ability of the enantiomers of the sample to differentially interact with a chiral phase that is part of the separation system.
The chiral phase can be embodied in a variety of ways. In chromatography, the chiral phase is conventionally part of the stationary phase, or column. In both GC and LC, a wide variety of chiral columns are available. The adsorption of the enantiomers by the stationary phase is the sum of both achiral and chiral interactions. The achiral interactions might include ionic, hydrogen bonding, and hydrophobic adsorption. The chiral interactions are derived from the spatial relationship of the achiral interactions. The energy difference contributed by this chiral interaction is the basis for the chiral separation.
The efficiency of the current generation of chiral chromatographic systems is generally low, thus the difference in the free energy of the interaction between the chiral modifier and the enantiomers must be relatively large in order to gain adequate resolution. This large energy difference requirement contributes to the low efficiency of many chiral HPLC systems (5000 to 10000 plates), and the tailing peaks observed on many chiral columns. This large energy difference requirement also prevents chiral HPLC columns from being of general use. Currently, chiral HPLC columns are selective for small classes of compounds, so more than fifty chiral phases have been commercialized. In this environment, method development is highly empirical and very tedious. Chiral separations per se have little novelty today, the challenge being to create systems which separate larger classes of enantiomers or provide easier method development.
In conventional gel electrophoresis, a chiral phase may be created by adding a chiral modifier to the gel buffer, or by covalent attachment of the modifier to the gel matrix (Barton, J. K., J. Biomolecular Structure and Dynamics, 1983, (1) 621-632). The separation occurs, as in chromatography, through a differential interaction of the individual enantiomers with the chiral phase. In gel electrophoresis, this results in a change in the overall electrophoretic mobilities of the two chiral molecules. As the two enantiomers move through the gel at different velocities, the separation is effected. Because the presence of the gel severely reduces bulk fluid movement, the osmotic flow that is commonly found in electrophoretic separations is minimized. Therefore, the final chiral separation is due largely to the change in electrophoretic mobility of the two enantiomers.
One of the major potential advantages of CE for chiral separations is the relatively high efficiency of the technique. This high efficiency permits the use of chiral modifiers that create only a small difference in free energies between the two enantiomers and the modifier. In CE the chiral phase is generally added to the supporting electrolyte. As in conventional gel electrophoresis, the two enantiomers bind differentially to the chiral modifier, resulting in a change in the electrophoretic mobilities. The resulting difference between the mobilities of the two enantiomers results in their separation. Although electroosmotic flow may sometimes be present in CE separations, it generally does not contribute significantly to the quality of the chiral separation.
Chiral CE separations were first disclosed in 1985 (Zare, R. N. and Gassmann, E. U.S. Pat. No. 4,675,300 Jun. 23, 1987), and a few samples have now been analyzed. However, the present art does not allow useful separations of a wise variety of compounds--it suffers from the same limitations as chiral HPLC separations; tedious method development and narrow selectivity. All separation shave been accomplished through the addition of a chiral modifier to the supporting electrolyte. The kind of chiral modifiers used fall into three categories. The first if the use of amino acid/metal complexes (Zare, R. N. and Gassmann, E. U.S. Pat. No. 4,675,300, Jun. 23, 1987; Gassmann, E., Kuo, J. E. and Zare, R. N. Science, 1985, (230) 813-814; Gozel, P., Gassmann, E., Michelsen, H and Zare, R. N. Analytical Chemistry, 1987, (59) 44-49). This type of complex is highly water soluble, and works well for chiral amino acid separations. This system has previously been shown to work using LC as well as gel electrophoresis for the same sample set. The second category of chiral modifiers is a class of carbohydrates called cyclodextrins (Guttmann, A., Paulus, A., Cohen, A. S., Grinberg, N. and Karger, B. L. Journal of Chromatography, 1988, (448) 41-53; Fanali, S. Journal of Chromatography, 1989, (474) 441-446). These modifiers are also highly water soluble, and are also employed extensively in LC. The third category of chiral modifiers are chiral detergents (Cohen, A. S., Paulus, A. and Karger, B. L. Chromatographia, 1987, (24) 15-24; Dobashi, A., Ono, T., Hara, S. and Yamaguchi, J. Analytical Chemistry, 1989, (61) 1984-1986; Terabe, S., Shibata, M. and Miyashita, Y. Journal of Chromatography, 1989, (480) 403-411).
The chiral detergent (S)-N,N-didecylalanine has been used with sodium dodecyl sulfate micelles and copper complexation to effect chiral separations (Cohen, A. S., Paulus, A. and Karger, B. L. Chromatographia, 1987, (24) 15-24). The chiral separation mechanism is based on ligand exchange as in the system reported by Zare, supra. The use of chiral detergents in the supporting electrolyte above their critical micelle concentration is art that has been practiced both with the use of sodium dodecanoylvaline (Dobashi, A., Ono, T., Hara, S. and Yamaguchi, J. Analytical Chemistry, 1989, (61) 1984-1986; Dobashi, A., Ono, T., Hara, S. and Yamaguchi, J. Journal of Chromatography, 1989, (480) 413-420), as well as with the use of bile salts (Terabe, S., Shibata, M. and Miyashita, Y. Journal of Chromatography, 1989, (48) 403-411). The micelle formed from these detergents is a structure composed of many individual detergent molecules. The inside of the micelle is generally a hydrophobic environment, much like the hydrophobic layer of a reversed phase chromatographic packing. The outside of the micelle presents hydrophilic groups that are often ionic, resulting in water solubility of the micelle particle. Somewhere in the surfactant there is a chiral center or centers which confer chirality to the micellar environment.
The present art of CE and in particular MEKC is similar to the HPLC art in that none of the systems have been shown to have broad applicability. Zare et al. have shown amino acid separations based on copper complexes which are well known from both chiral electrophoretic separations and chiral HPLC separations and are not useful for most other types of compounds. Cyclodextrin separations are restricted to molecules that can interact with the cyclodextrin cavity. Chiral MEKC separations have not been shown for acidic compounds nor for a range of compounds of a given class. Sodium dodecanoylvaline, as reported by Hara, has allowed chiral separations of neutral amino acid derivatives as well as a few other neutral compounds. Successful separation of organic bases or acids has not been demonstrated with this chiral surfactant. Bile salts have only been able to show chiral separation of analytes with very rigid structures containing fused ring systems.